Field of the Invention
The present invention relates to a mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide, to a method to produce the mutant microbial host cell and to a method to produce a compound of interest using said mutant microbial host cell.
Description of Related Art
Different host cell types may be used for different industrial purposes. For example: mammalian cell lines are used for antibody production; fungal cells are preferred organisms for production of polypeptides and secondary metabolites; bacterial cells are preferred for small metabolite and antibiotic production; and plant cells are preferred for taste and flavor compounds.
Recombinant techniques are widely employed for optimization of the productivity of such host cells and/or the processes in which they are used. This can involve a multitude of options.
Some techniques will aim at the over expression of a gene of interest coding for a compound of interest in the host cell. Gene expression can be modulated in several ways.
For example the gene of interest can be placed in the host cell under the expression control of a strong promoter, suitable for said cell. The latter can occur by introducing an expression cassette into the host cell, by plasmid- or vector-mediated transformation. Production of the compound of interest may then be achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained in the expression cassette. For example U.S. Pat. No. 5,722,8547 describes the use of DNA constructs used for transforming an Aspergillus to obtain expression therein of a polypeptide in which the DNA construct comprises an inducible promoter DNA for promoting transcription in Aspergillus and operably linked to a DNA coding for said polypeptide.
It is known that transcriptional activators are regulatory proteins facilitating the binding of RNA polymerase to a promoter controlling expression of a gene of interest. Gene expression can be modulated by for example using mutant host cells which produce a specific transcriptional activator in higher quantities, leading to increased expression of a gene of interest which is under the control of a promoter activated by said transcriptional activator. Such an approach is e.g. described in WO2006/040312, referring to the PrtT transcriptional activator and its use.
In yet an alternative approach gene expression can be improved by increasing the copy number of the gene of interest in the host cell used to express the gene. However the number of gene copies present in the host cell is a limiting factor as recombinant host cells comprising a high number of copies of a gene to be expressed may become unstable. A solution to this problem is given in WO9846772 which describes stable filamentous fungi comprising multiple substantially homologous DNA domains wherein in at least 2 of said domains an integrated copy of a recombinant DNA molecule coding for a compound of interest is present.
Yet other approaches aiming at improving the productivity of a compound of interest by a host cell can involve deletion or inactivation of competing pathways, changing compartmentalization of enzymes, increasing protein or metabolite secretion, increasing organelle content and the like.
Despite of advances in the understanding of expression of compounds of interests in host cells, there remains a need for methods to increase production of important compounds of interest on commercial or industrial scale. Surprisingly we have found that the down-regulation of the agsE gene in a microbial host cell, for example a filamentous fungal host cell expressing a compound of interest, e.g. en enzyme of interest, resulted in an increased production of said enzyme by said host cell.